Repetitive nucleotide sequences, such as direct or inverted repeats, are observed in many organisms. As an example, hundreds of thousands of microsatellite loci are distributed throughout the human genome and thus, occur statistically about once every 100,000 base pairs. A microsatellite locus is a region of genomic DNA that includes short tandem repeats in which the shortest repetitive units are typically from one to five nucleotides in length. Accordingly, a repetitive unit of a particular microsatellite locus is commonly referred to as a mono-, di-, tri-, tetra- or pentanucleotide repeat locus, as applicable. A given microsatellite locus typically includes between about 10 and 40 of these repetitive units in the tandem arrangement. Further, each microsatellite locus of normal genomic DNA for most diploid species, such as genomic DNA from mammalian species, includes two alleles at each locus. The two alleles can be identical to, or differ from, one another in length and may vary from one individual to the next.
Microsatellite instability (MSI), or replication error (RER), is an example of genomic instability that occurs in certain human neoplasms in which tumor cells have diminished abilities to accurately replicate their DNA. MSI is a common marker of an underlying functional inactivation of a human DNA mismatch repair (MMR) gene (Jeong et al. (2003) “Microsatellite instability and mutations in DNA mismatch repair genes in sporadic colorectal cancers,” Dis Colon Rectum. 46(8):1069-1077, Papadopoulos et al. (1994) “Mutations of a mutL homolog in hereditary colon cancer,” Science 263:1625-1629, Ghimenti et al. (1999) “Microsatellite instability and mismatch repair gene inactivation in sporadic pancreatic and colon tumours,” Br J Cancer. 80(1-2):11-16, Fishel et al. (1993) “The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer,” Cell 75:1027-1038, and Bronner et al. (1994) “Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer,” Nature 368:258-261, which are incorporated by reference). The functional loss of a MMR gene is thought to occur due to biallelic inactivation via coding region mutations, loss of heterozygosity (LOH), and/or promoter methylation (Goel et al. (2004) “Frequent inactivation of PTEN by promoter hypermethylation in microsatellite instability-high sporadic colorectal cancers,” Cancer Res. 64(9):3014-3021, Veigl et al. (1998) “Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers,” Proc Natl Acad Sci USA 95:8698-8702, and Piao et al. (2000) “Frequent frameshift mutations of RIZ in sporadic gastrointestinal and endometrial carcinomas with microsatellite instability,” Cancer Res. 60, 4701-4704, which are each incorporated by reference). Further, germline mutation of a MMR gene has been shown to be the autosomal dominant genetic defect in most hereditary nonpolyposis colon cancer (HNPCC) kindreds (Eshleman et al. (1995) “Microsatellite instability in inherited and sporadic neoplasms,” Curr Opin Oncol 7:83-89, which is incorporated by reference). A second mutation incurred by tumor cells in HNPCC individuals results in biallelic inactivation of the specific MMR gene, causing loss of accurate replication of microsatellite DNA in tumors (Hemminki et al. (1994) “Loss of the wild type MLH1 gene is a feature of hereditary nonpolyposis colorectal cancer,” Nat Genet 8:405-410, which is incorporated by reference). MSI is thus a marker of an underlying DNA mismatch repair defect and is also associated with enhanced mutation rates in coding DNA (Eshleman et al. (1995) “Increased mutation rate at the hprt locus accompanies microsatellite instability in colon cancer,” Oncogene 10:33-37 and Bhattacharyya et al. (1994) “Mutator phenotypes in human colorectal carcinoma cell lines,” Proc Natl Acad Sci USA 91:6319-6323, which is incorporated by reference). This mutator phenotype, which results from the MMR defect, causes both coding region base substitutions and frameshift mutations at direct repeats, each occurring at equal frequencies (Eshleman et al. (1996) “Diverse hypermutability of multiple expressed sequence motifs present in a cancer with microsatellite instability,” Oncogene 12:1425-1432, which is incorporated by reference), in addition to resulting in MSI. The generation of MMR defects and the resultant mutator phenotype is thought to be an early event in tumorigenesis (Parsons et al. (1993) “Hypermutability and mismatch repair deficiency in RER+ tumor cells,” Cell 75(6):1227-1236 and Shibata et al. (1994) “Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation,” Nat Genet 6:273-281, which are both incorporated by reference) and has been suggested to occur as early as the aberrant crypt focus stage (Augenlicht et al. (1996) “Evidence for genomic instability in human colonic aberrant crypt foci,” Oncogene 12:1767-1772, which is incorporated by reference).
Although implicating a germline defect in HNPCC families, MSI is also found in about 15 to 20% of sporadic colorectal cancers (Aaltonen et al. (1993) “Clues to the pathogenesis of familial colorectal cancer,” Science 260:812-816, which is incorporated by reference), where the finding also reflects an overall increase in genomic instability. The finding of MSI defects in tumors has also been associated with a better prognosis in stage-for-stage matched tumors (Thibodeau et al. (1993) “Microsatellite instability in cancer of the proximal colon,” Science 260:816-819 and Sankila et al. (1996) “Better survival rates in patients with MLH1-associated hereditary colorectal cancer,” Gastroenterology 110:682-687, which is incorporated by reference). Thus, it is clinically relevant to identify tumors with MSI not only to implicate germline MMR defects (HNPCC families), but also for prognostic stratification. While clinical (Bethesda guidelines (Rodriguez-Bigas et al. (1997) “A National Cancer Institute workshop on hereditary nonpolyposis colorectal cancer syndrome: meeting highlights and Bethesda guidelines,” J Natl Cancer Inst 89:1758-1762, which is incorporate by reference)) and histopathological features (Kim et al. (1994) “Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences,” Am J Pathol 145:148-156, which is incorporate by reference) may raise the suspicion that a colorectal tumor is microsatellite-unstable and perhaps has arisen in an HNPCC family, clinicopathological features are insufficient to diagnose the presence of MSI. Accordingly, molecular testing may be utilized to elucidate the MSI status of a clinically suspicious tumor (Boland et al. (1998) “A National Cancer Institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer,” Cancer Res 58:5248-5257, which is incorporated by reference).
In addition to colorectal tumors, MSI has also been associated with other types of cancer and other genetic disorders. To illustrate, these include among others, pancreatic carcinomas (Han et al. (1993) “Genetic instability in pancreatic cancer and poorly differentiated type of gastric cancer,” Cancer Res. 53:5087-5089), gastric carcinomas (Pan et al. (2004) “Detection of frameshift mutations of RIZ in gastric cancers with microsatellite instability,” World J Gastroenterol. 10(18):2719-2722, French et al. (2004) “Allelic imbalance of 8 p indicates poor survival in gastric cancer,” J Mol Diagn. 6(3):243-252, Rhyu et al. (1994) “Microsatellite instability occurs frequently in human gastric carcinoma,” Oncogene 9:29-32, and Han et al. (1993), supra), bladder cancer (Gonzalez-Zulueta et al. (1993) “Microsatellite instability in bladder cancer,” Cancer Res. 53:5620-5623), prostate carcinomas (Schoenberg et al. (1994) “Microsatellite mutation (CAG2418) in the androgen receptor gene in human prostate cancer,” Biochem. Biophys. Res. Commun. 198:74-80), lung cancers (Merlo et al. (1994) “Frequent microsatellite instability in primary small cell lung cancer,” Cancer Res. 54:2098-2101 and Shridhar et al. (1994) “Genetic instability of microsatellite sequences in many non-small cell lung carcinomas,” Cancer Res. 54:2084-2087), uterine carcinomas (Burks et al. (1994) “Microsatellite instability in endometrial carcinoma,” Oncogene 9:1163-1166 and Miyai et al. (2004) “Loss of heterozygosity analysis in uterine cervical adenocarcinoma,” Gynecol Oncol. 94(1):115-120) and breast cancer (Yang et al. (2004) “High-resolution 19p13.2-13.3 allelotyping of breast carcinomas demonstrates frequent loss of heterozygosity,” Genes Chromosomes Cancer 41(3):250-256 and Yee et al. (1994) “Microsatellite instability and loss of heterozygosity in breast cancer,” Cancer Res. 54:1641-1644). Other exemplary genetic orders thought to be related to microsatellite instability include, e.g., Huntington's disease (HD), dentatorubral and palidoluysian atrophy (DRPLA), spinobulbar and muscular atrophy (SBMA), myotonic dystrophy (DM), fragile X syndrome, FRAXE mental retardation and spinocerebellar ataxias (SCA) (Costa Lima et al. (2004) “Dynamic mutation and human disorders: the spinocerebellar ataxias (review),” Int J Mol Med. 13(2):299-302), Bruton X-linked agammaglobulinemia (XLA) (Allen et al. (1994) “Application of carrier testing to genetic counseling for X-linked agammaglobulinemia,” Am J Hum Genet. 54(1):25-35), Bloom syndrome (BS) (Foucault et al. (1996) “Stability of microsatellites and minisatellites in Bloom syndrome, a human syndrome of genetic instability,” Mutat Res. 362(3):227-236 and Kaneko et al. (1996) “Microsatellite instability in B-cell lymphoma originating from Bloom syndrome,” Int J Cancer. 69(6):480-483), craniofrontonasal syndrome (CFNS) (Feldman et al. (1997) “A novel phenotypic pattern in X-linked inheritance: craniofrontonasal syndrome maps to Xp22,” Hum Mol Genet. 6(11):1937-1941), and idiopathic pulmonary fibrosis (IPF) (Mori et al. (2001) “Microsatellite instability in transforming growth factor-beta 1 type II receptor gene in alveolar lining epithelial cells of idiopathic pulmonary fibrosis,” Am J Respir Cell Mol Biol. 24(4):398-404). All of the publications mentioned in this paragraph are incorporated by reference.
In view of the foregoing discussion, it is apparent that the analysis of repetitive nucleotide sequences, such as microsatellites has many diagnostic and prognostic applications among other uses. Among the limitations of pre-existing microsatellite analytical methodologies is the lack of rapid assays that can be performed in the order of minutes. For example, certain pre-existing MSI detection methods include amplifying microsatellite loci of interest by polymerase chain reaction (PCR), performing minisequencing reactions, and analyzing the products via gel electrophoresis. These complex, labor-intensive processes are not well suited to providing rapid results.